Protection against Fas receptor-mediated apoptosis in hepatocytes and nonparenchymal cells by a caspase-8 inhibitor in vivo: evidence for a postmitochondrial processing of caspase-8

Lymphocytes can kill target cells including hepatocytes during various inflammatory diseases by Fas receptor-mediated Apoptosis. Caspase-8 is activated at the receptor level, thereby initiating the processing of downstream effector caspases. The aim of this study was to investigate the time course of Caspase-8 activation and to evaluate the efficacy of the Caspase-8 inhibitor IETD-CHO in a model of Fas-induced Apoptosis in vivo. C3Heb/FeJ mice were treated with the anti-Fas antibody Jo-2 (0.6 mg/kg). Western blot analysis demonstrated increased cytochrome c in the cytosol (20 min), which was followed by the progressive activation of Caspase-3, -9 (40-120 min), and Caspase-8 (120 min). At 90 and 120 min, extensive hemorrhage was observed, indicating damage to sinusoidal lining cells. In addition, high plasma ALT levels (997 +/- 316 U/L) and histological evaluation indicated severe parenchymal cell injury. Parenchymal and nonparenchymal cells showed a similar increase in Caspase-3 activity and DNA fragmentation. Treatment with IETD-CHO (10 mg/kg) attenuated the increase in Caspase-3 activity and DNA fragmentation by 80-90% and completely prevented hemorrhage and parenchymal cell damage. IETD-CHO also prevented the early release of mitochondrial cytochrome c and the processing of Caspase-3, -8, and -9. Thus, our data support the hypothesis that Fas-mediated Apoptosis is dependent on Caspase-8 activation in hepatocytes and nonparenchymal cells. However, the bulk of procaspase-8 is processed late, suggesting that only a small amount of procaspase-8 may actually be activated at the Fas receptor. This initial signal may be amplified by further activation of Caspase-8 by effector caspases, i.e., after mitochondrial activation. Caspase-8 is a promising therapeutic target for inhibition of Fas-mediated Apoptosis.